To control HIV-1, it is thought that the induction of cell-mediated immunity may be critical for interrupting the establishment of infection. This proposal aims to identify a specific immunization protocol in which vaccination with combinations of DNA constructs encoding the chemokines RANTES, IL-8, or CTACK, and/or a novel B7DeltaC-domain costimulatory protein results in an enhanced antigen specific cell mediated immune response to the model HIV-1 Gag antigens. These novel adjuvants were selected because together they should be able to "target and trigger" (TNT) the immune response in vivo. The goal of this application will be to determine the best method for generating a TNT effect, and secondly, to determine the specific cell phenotypes of the responsible mediators. Several HIV-1 Gag DNA immunization strategies including adjuvant combinations with simultaneous intramuscular injections, sequential prime/boost regime, or alternative immunization timing schedules will be examined. Cellular and humoral immune responses will be measured quantitatively through the use of antibody titer ELISA, IFN-g ELISPOT, and Flow Cytometric staining (intracellular stains for IFN-g and TNF-a, and surface staining for the T cell markers CD45RO, CD27, CCR7, CCR5, and MHC Class I and II Tetramers). Experiments will be carried out in a novel CHAD (transgenic for human MHC Class I and II) murine model system developed in our laboratory. In vivo expression and functional kinetics of antigen and adjuvant in muscle cells will be determined by an immunohistochemical assay, using the unique GeneGrip reporter plasmid encoding GFP. Taken together, this proposal aims to identify and characterize a unique TNT adjuvant combination for HIV-1 DNA vaccines resulting in enhanced antigen specific cell-mediated immune responses and improved HIV-1 DNA vaccine candidates. [unreadable] [unreadable]